US2009035791A1PendingUtilityA1

Neural Colony Forming Assay

51
Individually held — no corporate assignee on recordPriority: Sep 12, 2003Filed: Jul 17, 2008Published: Feb 5, 2009
Est. expirySep 12, 2023(expired)· nominal 20-yr term from priority
Inventors:Brent ReynoldsSharon A. Louis
G01N 33/56966G01N 33/5073
51
PatentIndex Score
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Claims

Abstract

A neural colony forming cell (NCFC) assay is described. The assay allows one to distinguish neural stem cells from neural progenitor cells. In one embodiment, the present invention provides a method for identifying neural stem cells or neural progenitor cells comprising: (a) suspending neural cells in a semi-solid medium which supports the growth of neural cells; (b) plating the cells in the semi-solid medium at a density that allows for the production of colonies; (c) culturing the plated cells until size differences can be discerned between the colonies; and (d) estimating colony size wherein the larger colonies are likely produced by neural stem cells and wherein the small colonies are likely produced by neural progenitor cells In alternate embodiments, NSC can be distinguished from neural progenitor cells by determining the morphology or antigen expression of the colonies.

Claims

exact text as granted — not AI-modified
1 . A method for identifying neural stem cells or neural progenitor cells comprising:
 (a) suspending neural cells in a semi-solid medium which supports the growth of neural cells;   (b) plating the cells in the semi-solid medium at a cell density that allows for the production of colonies;   (c) culturing the plated cells until size differences can be discerned between the colonies; and   (d) estimating colony size wherein the larger colonies are likely produced by neural stem cells and wherein the smaller colonies are likely produced by neural progenitor cells.   
     
     
         2 . A method for identifying neural stem cells or neural progenitor cells comprising:
 (a) suspending neural cells in a semi-solid medium which supports the growth of neural cells;   (b) plating the cells in the semi-solid medium at a density that allows for the production of colonies;   (c) culturing the plated cells until colonies are formed; and   (d) determining the morphology of the colonies wherein the presence of undulated colonies indicates that the colonies are likely produced by neural stem cells and wherein the presence of colonies with a smooth periphery indicates that the colonies are likely produced by neural progenitor cells.   
     
     
         3 . A method for identifying neural stem cells or neural progenitor cells comprising:
 (a) suspending neural cells in a semi-solid medium which supports the growth of neural cells;   (b) plating the cells in the semi-solid medium at a density that allows for the production of colonies;   (c) culturing the plated cells until colonies are formed; and   (d) determining the antigen expression of the colonies wherein the presence of markers associated with undifferentiated cells indicates that the colonies are likely produced by neural stem cells and wherein the presence of markers associated with differentiated cells indicates that the colonies are likely produced by neural progenitor cells.   
     
     
         4 . A method according  claim 1  wherein the neural cells are mammalian. 
     
     
         5 . A method according to  claim 4  wherein the neural cells are from human, rat or mouse. 
     
     
         6 . A method according to  claim 1  wherein the neural cells are from primary CNS tissue or cultured neurospheres. 
     
     
         7 . A method according to  claim 1  wherein neural stem cells with high proliferative potential are discriminated from NSC with low proliferative potential. 
     
     
         8 . A method according to  claim 1  wherein the semi-solid medium is collagen based. 
     
     
         9 . A method according to  claim 1  wherein the semi-solid medium is methylcellulose based. 
     
     
         10 . A method according to  claim 1  wherein the neural cells are diluted in a culture medium prior to step (a). 
     
     
         11 . A method according to  claim 10  wherein a cytokine which promotes the growth of specific types of neural stem cells and neural progenitor cells is added to the medium. 
     
     
         12 . A method according to  claim 10  wherein a hormone which promotes the growth of specific types of neural stem cells and neural progenitor cells is added to the medium. 
     
     
         13 . A method according to  claim 12  wherein the culture medium is serum free. 
     
     
         14 . A method according to clam  1  wherein the cells are plated in step (b) at a density of about 1000 to 25,000 cells per 35 mm culture dish. 
     
     
         15 . A method according to clam  1  wherein the cells are cultured in step (c) for about 10 to about 28 days. 
     
     
         16 . A method according to  claim 1  wherein in step (d) a colony size of greater than 2.0 mm indicates neural stem cells. 
     
     
         17 . A method according to  claim 1  wherein in step (d) a colony size of less than or equal to 2.0 mm indicates neural progenitor cells. 
     
     
         18 . A method according to  claim 3  wherein the antigen expression of the colonies is determined using an in situ immunostaining protocol. 
     
     
         19 . A method according to  claim 3  wherein the antigen expression of the colonies is determined using an immunostaining protocol of plucked or excised neural stem or progenitor cell colonies. 
     
     
         20 . A method according to  claim 18  wherein the immunostaining uses an antibody directed against undifferentiated cells. 
     
     
         21 . A method according to  claim 20  wherein the antibody binds nestin, sox1, sox2, musashi or LexA/SSEA-1. 
     
     
         22 . A method according to  claim 18  wherein the immunostaining uses an antibody directed against cell markers specific to neural cell lineages. 
     
     
         23 . A method according to  claim 22  wherein the antibody binds to Beta-Tubulin, glial fibrillary acidic protein (GFAP), O4 or myelin basic protein (MBP). 
     
     
         24 . A use of an assay according to  claim 1  for testing potential therapeutic compositions. 
     
     
         25 . A use of an assay according to  claim 1  for diagnostic purposes. 
     
     
         26 . A use of an assay according to  claim 1  to assess the effects of agents in the environment. 
     
     
         27 . A use of an assay according to  claim 2  for testing potential therapeutic compositions. 
     
     
         28 . A use of an assay according to  claim 2  for diagnostic purposes. 
     
     
         29 . A use of an assay according to  claim 2  to assess the effects of agents in the environment. 
     
     
         30 . A use of an assay according to  claim 3  for testing potential therapeutic compositions. 
     
     
         31 . A use of an assay according to  claim 3  for diagnostic purposes. 
     
     
         32 . A use of an assay according to  claim 3  to assess the effects of agents in the environment.

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